The assay was according to the lipid-binding analysis method by Munnik and Wierzchowiecka (2013) (link). Ceremides, ganglioside GM3, galactocerebrosides, sulfatide, D-sphingosine, L-α-phosphatidylinositol and L-phosphatidylinositol (Sigma-Aldrich) were suspended in resuspension buffer (CHCl3/MeOH/H2O=20:9:1, v/v), and diluted to gradient concentration by spotting buffer (CHCl3/MeOH/50 mM HCl/Ponceau S=250:500:200:2, v/v). The lipids were spotted on PVDF membrane strips and dried at room temperature in the dark. The membranes were blocked in fat blot buffer (50 mM Tris–HCl, 150 mM NaCl, pH 7.5) containing 3% fatty acid-free BSA for 1 h at room temperature, and incubated with SMMC-7721 cell lysates (protein 5 μg/ml) in blocking buffer-0.1% Tween overnight at 4°C. After three washes with fat blot buffer-0.1% Tween, the membranes were incubated with anti-SIN3B antibody and goat anti-rabbit IgG HRP secondary antibody (Jackson ImmunoReseach Lab, Inc.). The blots were visualized using Immunobilon Western (Millipore).
Immunobilon western
Manufactured by Merck Group
Sourced in United States
Immunobilon Western is a type of membrane used in Western blot analysis. It is designed for the efficient transfer and immobilization of proteins from polyacrylamide gels to a membrane support for subsequent detection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
D sphingosine, by Merck Group (1 mentions)
L phosphatidylinositol, by Merck Group (1 mentions)
L α phosphatidylinositol, by Merck Group (1 mentions)
Ero1 lα, by Cell Signaling Technology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
Imagequant tl 7, by GE Healthcare (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
2 protocols using immunobilon western
1
Lipid-binding Analysis in SMMC-7721 Cells
2
Western Blot Analysis of Cellular Stress Responses
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Western blot analysis was performed as previously described32 (link). The culture medium was replaced with a new medium when the HepG2 cells were 70% confluent, then, the cells were exposed to various concentrations of TNT for 24 h. Subsequently, the cells were washed twice with PBS and then lysed. From each sample, 50 μg protein was subjected to 10% SDS polyacrylamide gel electrophoresis, transferred to PVDF membranes, and blocked with 5% skim milk at room temperature for 1 h. After blocking, the membrane was incubated with antibodies against pro-caspase-9, pro-caspase-3, cleaved-caspase-9, cleaved-caspase-3, β-actin, calnexin, PDI, BIP, IRE1-α, PERK, Ero1-Lα, CHOP, Bax, Bcl-2, phosphorylation of IRE1, phosphorylation of PERK and phosphorylation of eIF2α (Cell Signaling, USA) for 1 h. Then, the membranes were washed with 0.1% PBST (PBS and 0.05% Tween 20) and incubated with secondary antibodies conjugated to horseradish peroxidase for 1 h. Bands were detected after chemiluminescent HRP (Immunobilon Western, Millipore) was added. The band density was measured with ImageQuant-TL7.0 software (GE Healthcare).
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